Apoptotic cell suicide is a fundamentally important biological process that is required to maintain the integrity and homeostasis of multicellular organisms. Inappropriate apoptosis, however, underlies the etiology of many of the most intractable of human diseases. In only the last few years, many of the molecules that participate in a conserved biochemical pathway that mediates the highly ordered process of apoptotic cell suicide have been identified. At the heart of this pathway are a family of cysteine proteases, the xe2x80x98caspasesxe2x80x99, that are related to mammalian interleukin-1xcex2 converting enzyme (ICE/caspase-1) and to CED-3, the product of a gene that is necessary for apoptotic suicide in the nematode C. elegans (Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The role of these proteases in cell suicide is to disable critical homeostatic and repair processes as well as to cleave key structural components, resulting in the systematic and orderly disassembly of the dying cell.
The central importance of caspases in these processes has been demonstrated with both macromolecular and peptide-based inhibitors (which prevent apoptosis from occurring in vitro and in vivo) as well as by genetic approaches. Inhibition of apoptosis via attenuation of caspase activity should therefore be useful in the treatment of human diseases where inappropriate apoptosis is prominent or contributes to disease pathogenesis. Caspase inhibitors would thus be useful for the treatment of human diseases including, but not limited to, acute disorders such as cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), spinal cord injury and organ damage during transplantation, sepsis, bacterial meningitis, chronic disorders such as neurodegenerative diseases (e.g. Alzheimer""s, polyglutamine-repeat disorders, Down""s, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging.
Thirteen caspases have so far been identified in human cells. Each is synthesized as a catalytically dormant proenzyme containing an amino-terminal pro-domain followed by the large and small subunits of the heterodimeric active enzyme. The subunits are excised from the proenzyme by cleavage at Asp-X junctions (Nicholson et al., 1997, Trends Biochem Sci 22:299-306). The strict requirement by caspases for Asp in the P1 position of substrates is consistent with a mechanism whereby proenzyme maturation can be either autocatalytic or performed by other caspases. The three dimensional crystal structures of mature caspase-1 and -3 show that the large subunit contains the principle components of the catalytic machinery, including the active site Cys residue which is harbored within the conserved pentapeptide motif, QACxG, and residues that stabilize the oxyanion of the tetrahedral transition state (Wilson et al., 1994, Nature 370:270-75; Walker et al., 1994, Cell 78:342-52; Rotonda et al., 1996, Nat Struct Biol 3:619-25). Both subunits contribute residues which stabilize the P1 Asp of substrates while the small subunit appears to contain most of the determinants that dictate substrate specificity and, in particular, those which form the specificity-determining S4 subsite. One distinctive feature of these proteases is the absolute requirement for an aspartic acid residue in the substrate P1 position. The carboxylate side chain of the substrate P1 Asp is tethered by four residues in caspase-1 (Arg179, Gln238 from p20 and Arg341, Ser347 from p10) that are absolutely conserved in all caspase family members. Catalysis involves a typical cysteine protease mechanism involving a catalytic dyad, composed of His237 and Cys285 (contained within an absolutely conserved QACxG pentapeptide) and an xe2x80x98oxyanion holexe2x80x99 involving Gly238 and Cys285. Inhibitors bind, however, in an unexpected non-transition state configuration (which raises important considerations for inhibitor design) with the oxyanion of the thiohemiacetal being stabilized by the active site His237.
Members of the caspase family can be divided into three functional subgroups based on their substrate specificities which have been defined by a positional-scanning combinatorial substrate approach. The principle effectors of apoptosis (group II caspases, which include caspases-2, -3 and -7 as well as C. elegans CED-3) have specificity for [P4]DExD[P1], a motif found at the cleavage site of most proteins known to be cleaved during apoptosis. On the other hand, the specificity of group III caspases (caspases-6, -8, -9 and -10, as well as CTL-derived granzyme B) is [P4](I,V,L)ExD[P1] which corresponds to the activation site at the junction between the large and small subunits of other caspase proenzymes including group II (effector) family members. This and other evidence indicates that group III caspases function as upstream activators of group II caspases in a proteolytic cascade that amplifies the death signal. The role of group I caspases (caspases-1, -4 and -5) appears to be to mediate cytokine maturation and their role in apoptosis, if any, has not been substantiated.
A tetrapeptide corresponding to the substrate P4-P1 residues is sufficient for specific recognition by caspases and as a consequence has formed the basis for inhibitor design. In addition to the requirement for a P1 Asp, the P4 residue in particular appears to be most important for substrate recognition and specificity. Caspase-1, for example, prefers a hydrophobic residue such as Tyr in P4 (which corresponds to its YVHD cleavage site within proIL-1xcex2) whereas caspase-3 (and other group II enzymes) has a preference for an anionic Asp residue (which corresponds to the DXXD cleavage sites within most polypeptides that are cleaved by these enzymes during apoptosis). Peptide aldehydes, nitriles and ketones are potent reversible inhibitors of these proteases while compounds that form thiomethylketone adducts with the active site cysteine (e.g. peptide (acyloxy)methylketones) are potent irreversible inhibitors. For example, the tetrapeptide aldehyde Ac-YVAD-CHO (which was designed to mimic the YVHD caspase-1 recognition sequence within proIL-1xcex2) is a potent inhibitor of caspase-1 (Ki less than 1 nM) but a poor inhibitor of caspase-3 (Ki=12 xcexcM) (Thornberry et al., 1992, Nature 356:768-74). In contrast, the Ac-DEVD-CHO tetrapeptide aldehyde (which was designed to mimic the caspase-3 recognition site) is a very potent inhibitor of caspase-3 (Ki less than 1 nM) although it is also a weaker but reasonable inhibitor of caspase-1, presumably owing to promiscuity in the S4 subsite of this enzyme (Nicholson et al., 1995, Nature 376:37-43).
Several features plague these peptide-derived inhibitors as a platform for drug design. In addition to their poor metabolic stability and poor membrane permeability, the slow-binding time-dependent inhibition of activity (e.g. kon caspase-1: Ac-YVAD-CHO=3.8xc3x97105 Mxe2x88x921sxe2x88x921; kon caspase-3 Ac-DEVD-CHO=1.3xc3x97105 Mxe2x88x921sxe2x88x921) precludes them from the rapid inhibition characteristics that may be necessary to abolish enzymatic activity in vivo. The present invention describes the resolution of these issues with the discovery of a novel series of non-peptidyl caspase inhibitors containing a pyrazinone core.
A compound represented by formula I: 
or a pharmaceutically acceptable salt, ester, N-oxide or hydrate thereof wherein:
R1 is selected from the group consisting of: OH, C1-6alkyl, HET, Aryl, C1-6alkoxy, NH2, NHC1-6alkyl, N(C1-6alkyl)2, C1-6alkylC(O), C1-6alkylS(O)y, Aryl-S(O)y, HETxe2x80x94S(O)y wherein y is 0, 1 or 2, Aryl-C(O) and HET-C(O),
the alkyl and alkyl portions of which being optionally substituted with 1-2 members selected from the group consisting of: OH, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4-acyl;
Aryl represents a C6-14aromatic 1-3 ring system optionally substituted with 1-3 members selected from OH, C1-6alkyl, OC1-6alkyl, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CF3, CO2H and C1-4acyl;
Aryl 1 represents a C6-14 membered aromatic ring system having 1-3 rings and optionally substituted with 1-3 members selected from the group consisting of: OH, HET, halo, NH2, NHCH3, N(CH3)2, CO2H and C1-4-acyl;
HET represents a 5 to 15 membered aromatic, partially aromatic or non-aromatic ring system, containing 1-4 heteroatoms selected from O, S and N, and optionally substituted with 1-2 oxo groups and 1-3 groups selected from halo, C1 4alkyl, C, 4alkoxy, CF3 and C1-4acyl;
Ra and Rb independently represent a member selected from the group consisting of: H, Aryl, C, 6alkyl optionally substituted by 1-3 of halo, OR4, SR4 and C5-7cycloalkyl optionally containing one heteroatom selected from O, S and NR5,
or in the alternative, Ra and Rb are taken in combination and represent a non-aromatic carbocyclic 4-7 membered ring, optionally containing one heteroatom selected from O, S and NR5;
R4 is selected from the group consisting of: H, C1-5alkyl, Aryl and Aryl-C1-4alkyl optionally substituted with 1-2 groups selected from halo and C1-4alkyl;
R5 is H, C1-4alkyl or C1-4acyl;
Rc and Rd each independently represents a member selected from the group consisting of: H, C1-6alkyl and Aryl, or in the alternative, Rc and Rd are taken in combination and represent a non-aromatic carbocyclic ring of 3-7 members, optionally containing one heteroatom selected from O, S and NR5;
n is an integer from 0-6 inclusive;
R2 represents H, halo or C1-6alkyl;
R3 represents H, C1-6alkyl, Aryl, HET, C1-6alkylSR6, C1-6alkylOR6, C1-6alkylOC(O)R7 or C1-6alkylNR8R9;
R6 represents C1-6alkyl, Aryl, HET or Aryl-C1-6alkyl, said alkyl and the alkyl portions being optionally substituted with 1-3 members selected from the group consisting of: OH, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4acyl;
R7 represents C1-8alkyl, Aryl or HET;
R8 and R9 independently represent H, C1-10alkyl, Aryl, HET, C1-6alkylN(C1-6alkyl)0-2, Aryl-C1-6alkyl , C1-6alkylOH, or C1-6alkylOC1-6alkyl , or R8 and R9 are taken in combination with the nitrogen atom to which they are attached and represent a 3-10 membered ring system containing 1-4 heteroatoms selected from O, S, N and optionally substituted with 1-2 oxo groups, and 1-3 groups selected from C1-6alkyl, HET, CO2Rc and C(O)N(Rc)2,
said alkyl and alkyl portions being optionally substituted with 1-3 groups selected from halo, C1-3alkyl, hydroxyC1-3alkyl, C1-3alkoxy, C1-3alkoxyC1-3alkyl and Aryl1, and
R10 represents H, C1-20alkyl, aryl or HET, with aryl and HET as previously described.
The invention also encompasses a pharmaceutical composition comprising a compound of formula I in combination with a pharmaceutically acceptable carrier.
The present invention relates to a compound represented by formula I: 
or a pharmaceutically acceptable salt, ester, N-oxide or hydrate thereof wherein:
R1 is selected from the group consisting of: OH, C1-6alkyl, HET, Aryl, C1-6alkoxy, NH2, NHC1-6alkyl, N(C1-6alkyl)2, C1-6alkylC(O), C1-6alkylS(O)y, Aryl-S(O)y, HETxe2x80x94S(O)y wherein y is 0, 1 or 2, Aryl-C(O) and HETxe2x80x94C(O),
the alkyl and alkyl portions of which being optionally substituted with 1-2 members selected from the group consisting of: OH, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4-acyl;
Aryl represents a C6-14aromatic 1-3 ring system optionally substituted with 1-3 members selected from OH, C1-6alkyl, OC1-6alkyl, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CF3, CO2H and C1-4acyl;
Aryl1 represents a C6-14 membered aromatic ring system having 1-3 rings and optionally substituted with 1-3 members selected from the group consisting of: OH, HET, halo, NH2, NHCH3, N(CH3)2, CO2H and C1-4-acyl;
HET represents a 5 to 15 membered aromatic, partially aromatic or non-aromatic ring system, containing 1-4 heteroatoms selected from O, S and N, and optionally substituted with 1-2 oxo groups and 1-3 groups selected from halo, C1-4alkyl, C1-4alkoxy, CF3 and C1-4acyl;
Ra and Rb independently represent a member selected from the group consisting of: H, Aryl, C1-6alkyl optionally substituted by 1-3 of halo, OR4, SR4 and C5-7cycloalkyl optionally containing one heteroatom selected from O, S and NR5,
or in the alternative, Ra and Rb are taken in combination and represent a non-aromatic carbocyclic 4-7 membered ring, optionally containing one heteroatom selected from O, S and NR5;
R4 is selected from the group consisting of: H, C1-5alkyl, Aryl and Aryl-C1-4alkyl optionally substituted with 1-2 groups selected from halo and C1-4alkyl;
R5 is H or C1-4alkyl;
Rc and Rd each independently represents a member selected from the group consisting of: H, C1-6alkyl and Aryl, or in the alternative, Rc and Rd are taken in combination and represent a non-aromatic carbocyclic ring of 3-7 members, optionally containing one heteroatom selected from O, S and NR5;
n is an integer from 0-6 inclusive;
R2 represents H, halo or C1-6alkyl;
R3 represents H, C1-6alkyl, Aryl, HET, C1-6alkylSR6, C1-6alkylOR6, C1-6alkylOC(O)R7 or C1-6alkylNR8R9;
R6 represents C1-6alkyl, Aryl, HET or Aryl-C1-6alkyl, said alkyl and the alkyl portions being optionally substituted with 1-3 members selected from the group consisting of: OH, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4acyl;
R7 represents C1-8alkyl, Aryl or HET;
R8 and R9 independently represent H, C1-10alkyl, Aryl, HET, C1-6alkylN(C1-6alkyl)0-2, Aryl-C1-6alkyl , C1-6alkylOH, or C1-6alkylOC1-6alkyl , or RR9 are taken in combination with the nitrogen atom to which they are attached and represent a 3-10 membered ring system containing 1-4 heteroatoms selected from O, S, N and optionally substituted with 1-2 oxo groups, and 1-3 groups selected from C1-6alkyl, HET, CO2Rc and C(O)N(Rc)2,
said alkyl and alkyl portions being optionally substituted with 1-3 groups selected from halo, C1-3alkyl, hydroxyC1-3alkyl, C1-3alkoxy, C1-3alkoxyC1-3alkyl and Aryl1, and
R10 represents H, C1-20alkyl, aryl or HET, with aryl and HET as previously described.
More particularly, the present invention relates to a compound represented by formula Ixe2x80x2: 
or a pharmaceutically acceptable salt, ester, N-oxide or hydrate thereof wherein:
R1 is selected from the group consisting of: OH, C1-6alkyl, HET, Aryl, C1-6alkoxy, NH2, NHC1-6alkyl, N(C1-6alkyl)2, C1-6alkylC(O), C1-6alkylS(O)Y, Aryl-S(O)y, HETxe2x80x94S(O)y wherein y is 0, 1 or 2, Aryl-C(O) and HETxe2x80x94C(O),
the alkyl and alkyl portions of which being optionally substituted with 1-2 members selected from the group consisting of: OH, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4-acyl;
Aryl represents a C6-14aromatic 1-3 ring system optionally substituted with 1-3 members selected from OH, C1-6alkyl, OC1-6alkyl, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CF3, CO2H and C1-4acyl;
Aryl1 represents a C6-14 membered aromatic ring system having 1-3 rings and optionally substituted with 1-3 members selected from the group consisting of: OH, HET, halo, NH2, NHCH3, N(CH3)2, CO2H and C1-4-acyl;
HET represents a 5 to 15 membered aromatic, partially aromatic or non-aromatic ring system, containing 1-4 heteroatoms selected from O, S and N, and optionally substituted with 1-2 oxo groups and 1-3 groups selected from halo, C1-4alkyl, C1-4alkoxy, CF3 and C1-4acyl;
Ra and Rb independently represent a member selected from the group consisting of: H, Aryl, C1-6alkyl optionally substituted by 1-3 of halo, OR4, SR4 and C5-7cycloalkyl optionally containing one heteroatom selected from O, S and NR5,
or in the alternative, Ra and Rb are taken in combination and represent a non-aromatic carbocyclic 4-7 membered ring, optionally containing one heteroatom selected from O, S and NR5;
R4 is selected from the group consisting of: H, C1-5alkyl, Aryl and Aryl-C1-4alkyl optionally substituted with 1-2 groups selected from halo and C1-4alkyl;
R5 is H or C1-4alkyl;
Rc and Rd each independently represents a member selected from the group consisting of: H, C1-6alkyl and Aryl, or in the alternative, Rc and Rd are taken in combination and represent a non-aromatic carbocyclic ring of 3-7 members, optionally containing one heteroatom selected from O, S and NR5;
n is an integer from 0-6 inclusive;
R2 represents H, halo or C1-6alkyl;
R3 represents H, C1-6alkyl, Aryl, HET, C1-6alkylSR6, C1-6alkylOR6, C1-6alkylOC(O)R7 or C1-6alkylNR8R9;
R6 represents C1-6alkyl, Aryl, HET or Aryl-C1-6alkyl, said alkyl and the alkyl portions being optionally substituted with 1-3 members selected from the group consisting of: OH, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4acyl;
R7 represents C1-8alkyl, Aryl or HET;
R8 and R9 independently represent H, C1-10alkyl, Aryl, HET, C1-6alkylN(C1-6alkyl)0-2, Aryl-C1-6alkyl, C1-6alkylOH, or C1-6alkylOC1-6alkyl , or R8 and R9 are taken in combination with the nitrogen atom to which they are attached and represent a 3-10 membered ring system containing 1-4 heteroatoms selected from O, S, N and optionally substituted with 1-2 oxo groups, and 1-3 groups selected from C1-6alkyl, HET, CO2Rc and C(O)N(Rc)2,
said alkyl and alkyl portions being optionally substituted with 1-3 groups selected from halo, C1-3alkyl, hydroxyC1-3alkyl, C1-3alkoxy, C1-3alkoxyC1-3alkyl and Aryl1.
The invention also encompasses a pharmaceutical composition comprising a compound of formula I in combination with a pharmaceutically acceptable carrier.
The invention also encompasses a method of treating cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), type I diabetes, immune deficiency syndrome (including AIDS), cerebral and spinal cord trauma injury, organ damage during transplantation, alopecia, sepsis, bacterial meningitis, aging, Parkinson""s disease, Alzheimer""s disease, Down""s syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders, comprising administering to a mammalian patient in need of such treatment an effective amount of a compound of formula I.
Alkyl as used herein means linear, branched or cyclic structures and combinations thereof, containing one to twenty carbon atoms unless otherwise specified. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, eicosyl, 3,7-diethyl-2,2-dimethyl-4-propylnonyl, cyclopropyl, cyclopentyl, cycloheptyl, adamantyl, cyclododecylmethyl, 2-ethyl-1-bicyclo[4.4.0]decyl and the like.
Alkylcarbonyl signifies groups having the formula xe2x80x94C(O)-alkyl, wherein alkyl is defined as above.
Alkylsulfonyl signifies groups having the formula xe2x80x94S(O)2-alkyl, wherein alkyl is defined as above.
Fluoroalkyl means linear, branched or cyclic alkyl groups and combinations thereof, of one to ten carbon atoms, in which one or more hydrogen but no more than six is replaced by fluorine. Examples are xe2x80x94CF3, xe2x80x94CH2CH2F, and xe2x80x94CH2CF3 and the like.
Alkoxy means alkoxy groups of one to ten carbon atoms of a straight, branched or cyclic configuration. Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, and the like.
Alkoxycarbonyl signifies groups having the formula xe2x80x94C(O)-alkoxy, wherein alkoxy is defined as above.
Alkylthio means alkylthio groups of one to ten carbon atoms of a straight, branched or cyclic configuration. Examples of alkylthio groups include methylthio, propylthio, isopropylthio, etc. By way of illustration, the propylthio group signifies xe2x80x94SCH2CH2CH3.
Aryl is a 1-3 ring aromatic group containing 6-14 carbon atoms. Examples include phenyl, naphthyl, phenanthrenyl and the like. Ring system refers to single rings as well as 2-4 rings that are fused.
HET represents a 5 to 15 membered aromatic, partially aromatic or non-aromatic ring system, containing 1-4 heteroatoms selected from O, S and N, and optionally substituted with 1-2 oxo groups and 1-3 groups selected from halo, C1-4alkyl, C1-4alkoxy, CF3 and C1-4acyl. HET thus includes heteroaryl and heterocyclyl.
Heteroaryl is a heteroaromatic 5-15 membered group containing at least one heteroatom selected from O, S and N with up to 4 such heteroatoms being present in the ring system, e.g., pyridyl, furyl, thienyl, thiazolyl, isothiazolyl, imidazolyl, benzimidazolyl, pyrazinyl, pyrimidyl, quinolyl, isoquinolyl, benzofuryl, benzothienyl, pyrazolyl, indolyl, purinyl, isoxazolyl, oxazolyl, coumarinyl, benzocoumarinyl and the like.
Halo includes F, Cl, Br and I.
N-oxide refers to oxides of the N atoms in the HET groups.
For purposes of this specification, the following abbreviations have the indicated meanings:
Alkyl group abbreviations:
One subgroup of compounds that is of particular interest relates to compounds of formula I wherein R1 represents HET or Aryl,
said HET representing a 5 to 15 membered aromatic, partially aromatic or non-aromatic ring or ring system, containing from 1-4 heteroatoms selected from O, S and N, and optionally substituted with 1-2 groups selected from oxo, halo, C1-4alkyl, C1-4alkoxy and C1-4acyl, and
said Aryl being selected from phenyl and naphthyl, and being optionally substituted with 1-3 members selected from the group consisting of: OH, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CO2H and C1-4-acyl. Within this subset of compounds, all other variables are as originally defined.
More particularly, a subgroup that is of interest relates to compounds of formula I wherein R1 represents HET optionally substituted with 1-2 groups selected from oxo, halo, C1-4alkyl, C1-4alkoxy and C1-4acyl. Within this subset of compounds, all other variables are as originally defined.
Even more particularly, a subgroup that is of interest relates to compounds of formula I wherein R1 represents HET substituted with 1-2 groups selected from oxo, halo, C1-4alkyl, C1-4alkoxy and C1-4acyl. Within this subset of compounds, all other variables are as originally defined.
Even more particularly, a subgroup that is of interest relates to compounds of formula I wherein R1 represents HET selected from the group consisting of: pyridinyl, pyrazinyl, pyrrolyl, furanyl, pyrazolyl, imidazolyl, benzimidazolyl, oxathiazolyl, thiazolyl, benzothiazolyl, oxazolyl, pyrrazolyl, 1,2-diazolyl, 1,2,3- and 1,2,4-triazolyl, 1,2,4- and 1,2,5-oxadiazolyl, 1,2,4- and 1,2,5-thiadiazolyl, tetrazolyl, isoxazolyl, thienyl, azepinyl, pyrrolidinyl, piperidinyl, piperazinyl, optionally substituted with 1-2 groups selected from halo, C1-4alkyl and C1-4alkoxy. Within this subset of compounds, all other variables are as originally defined.
Another group of compounds that is of particular interest relates to compounds of formula I wherein R1 represents Aryl said Aryl being phenyl optionally substituted with 1-3 members selected from the group consisting of: OH, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CO2H and C1-4-acyl. Within this subset of compounds, all other variables are as originally defined.
Another group of compounds that is particular interest relates to compounds of formula I wherein Rc and Rd represent H, and n is an integer of from 0-3 inclusive. In particular, (RcRd)n represents methylene, ethylene or propylene.
Another group of compounds that is particular interest relates to compounds of formula I wherein Ra and Rb independently represent H or C1-6alkyl, optionally substituted with halo, OR4, SR4 or C5-7cycloalkyl optionally containing one heteroatom selected from O, S and NR5. Within this subset of compounds, all other variables are as originally defined.
More particularly, one of Ra and Rb represents H and the other represents C1-6alkyl. Within this subset of compounds, all other variables are as originally defined.
Even more particularly, one of Ra and Rb represents H and the other represents ethyl. Within this subset of compounds, all other variables are as originally defined.
Another group of compounds that is particular interest relates to compounds of formula I wherein R2 represents H or Halo. Within this subset of compounds, all other variables are as originally defined.
Another group of compounds that is particular interest relates to compounds of formula I wherein:
R3 is selected from the group consisting of H, C1-6alkyl, C1-6alkylSR6, and C1-6alkylNR8R9;
R6 represents C1-6alkyl, Aryl, HET or Aryl-C1-6alkyl, said alkyl, aryl, and the alkyl group and alkyl portions being optionally substituted with 1-3 members selected from the group consisting of: OH, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4acyl, and said HET being optionally substituted with 1-2 oxo groups and 1-3 groups selected from halo, C1-4alkyl, C1-4alkoxy, CF3 and C1-4acyl; and
R8 and R9 independently represent H, C10alkyl, Aryl, HET, C1-6alkylN(C1-6alkyl)0-2, Aryl-C1-6alkyl , C1-6alkylOH, or C1-6alkylOC1-6alkyl , or R8 and R9 are taken in combination with the nitrogen atom to which they are attached and represent a 3-10 membered ring system containing 1-4 heteroatoms selected from O, S, N and optionally substituted with 1-2 oxo groups, and 1-3 groups selected from C1-6alkyl, HET, CO2Rc and C(O)N(Rc)2,
said alkyl and alkyl portions being optionally substituted with 1-3 groups selected from halo, C1-3alkyl, hydroxyC1-3alkyl, C1-3alkoxy, C1-3alkoxyC1-3alkyl and Aryl1. Within this subset, all other variables are as originally defined.
More particularly, a group of compounds that is of interest relates to compounds of formula I wherein:
R3 is selected from the group consisting of: H, C1-6alkyl, C1-6alkylSR6 and C1-6alkylNR8R9;
R6 represents Aryl, HET or Aryl-C1-6alkyl, said alkyl, aryl, and the alkyl group and alkyl portions being optionally substituted with 1-3 members selected from the group consisting of: OH, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4acyl, and said HET being optionally substituted with 1-2 oxo groups and 1-3 groups selected from halo and C1-4alkyl; and
R8 and R9 independently represent H, C1-10alkyl, Aryl, HET, C1-6alkylN(C1-6alkyl)0-2, Aryl-C1-6alkyl or C1-6alkylOC1-6alkyl , or R8 and R9 are taken in combination with the nitrogen atom to which they are attached and represent a 3-10 membered ring system containing 1-4 heteroatoms selected from O, S, N and optionally substituted with 1-2 oxo groups, and 1-3 groups selected from C1-6alkyl, HET, CO2Rc and C(O)N(Rc)2,
said alkyl and alkyl portions being optionally substituted with 1-3 groups selected from halo, C1-3alkyl, C1-3alkoxyC1-3alkyl and Aryl1. Within this subset, all other variables are as originally defined.
Another subgroup of compounds that is of particular interest relates to compounds of formula I wherein R10 represents H, C1-8alkyl or aryl. Within this subset, all other variables are as previously described.
More particularly, the subgroup of compounds that is of particular interest relates to compounds of formula I wherein R10 is selected from the group consisting of: H, methyl, ethyl, isopropyl, t-butyl and phenyl. Within this subset, all other variables are as previously described.
Another subgroup of compounds that is of particular interest relates to compounds of formula I wherein n is 1-6. More particularly, the subgroup of particular interest relates to compounds of formula I wherein n is 1-3. Within this subset, all other variables are as previously described.
One subgroup of compounds that is of particular interest relates to compounds of formula I wherein:
R1 represents HET or Aryl, said HET representing a 5 to 15 membered aromatic, partially aromatic or non-aromatic ring or ring system, containing from 1-4 heteroatoms selected from O, S and N, and optionally substituted with 1-2 groups selected from oxo, halo, C1-4alkyl C1-4alkoxy and C1-4acyl, and said Aryl being selected from phenyl and naphthyl, and being optionally substituted with 1-3 members selected from the group consisting of: OH, Aryl1, HET, halo, NH2, NHCH3, N(CH3)2, CO2H and C1-4-acyl;
Rc and Rd represent H, and n is an integer of from 0-3 inclusive;
Ra and Rb independently represent H or C1-6alkyl optionally substituted with halo, OR4, SR4 or C57 cycloalkyl optionally containing one heteroatom selected from O, S and NR5;
R3 is selected from the group consisting of H, C1-6alkyl, C1-6alkylSR6, and C1-6alkylNR8R9;
R6 represents C1-6alkyl, Aryl, HET or Aryl-C1-6alkyl, said alkyl, aryl, and the alkyl group and alkyl portions being optionally substituted with 1-3 members selected from the group consisting of: OH, halo, NH2, NHCH3, N(CH3)2, CO2H, CF3 and C1-4acyl, and said HET being optionally substituted with 1-2 oxo groups and 1-3 groups selected from halo, C1-4alkyl, C1-4alkoxy, CF3 and C1-4acyl; and
R8 and R9 independently represent H, C1-10alkyl, Aryl, HET, C1-6alkylN(C1-6alkyl)0-2, Aryl-C1-6alkyl , C1-6alkylOH, or C1-6alkylOC1-6alkyl, or R8 and R9 are taken in combination with the nitrogen atom to which they are attached and represent a 3-10 membered ring system containing 1-4 heteroatoms selected from O, S, N and optionally substituted with 1-2 oxo groups, and 1-3 groups selected from C1-6alkyl, HET, CO2Rc and C(O)N(Rc)2,
said alkyl and alkyl portions being optionally substituted with 1-3 groups selected from halo, C1-3alkyl, hydroxyC1-3alkyl, C1-3alkoxy, C1-3alkoxyC1-3alkyl and Aryl1, and
R10 represents H, C1-8alkyl or aryl. Within this subset, all other variables are as originally defined.
Representative examples of compounds of formula I are found in Table 1 below.
The compounds described herein, and in particular, in Table 1, are intended to include salts, enantiomers, esters, N-oxides and hydrates, in pure form and as a mixture thereof. While chiral structures are shown below, by substituting into the synthesis schemes an enantiomer other than the one shown, or by substituting into the schemes a mixture of enantiomers, a different isomer or a racemic mixture can be achieved. Thus, all such isomers and mixtures are included in the present invention.
In another embodiment, the invention encompasses a method of treating or preventing a caspase-3 mediated disease or condition in a mammalian patient in need thereof, comprising administering to said patient a compound of formula I in an amount effective to treat or prevent said caspase-3 mediated disease or condition.
In another embodiment, the invention encompasses a method of treating cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), type I diabetes, immune deficiency syndrome (including AIDS), cerebral and spinal cord trauma injury, organ damage during transplantation, sepsis, bacterial meningitis, alopecia, aging, Parkinson""s disease, Alzheimer""s disease, Down""s syndrome, spinal muscular atrophy, multiple sclerosis and neurodegenerative disorders, comprising administering to a mammalian patient in need of such treatment an effective amount of a compound of formula I.
In another embodiment, the invention encompasses a method of treating acute disorders, including cardiac and cerebral ischemia/reperfusion injury (e.g. stroke), sepsis, bacterial meningitis, spinal cord injury and organ damage during transplantation, in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said acute disorder.
In another embodiment, the invention encompasses a method of treating chronic disorders, including neurodegenerative diseases (e.g. Alzheimer""s, polyglutamine-repeat disorders, Down""s, spinal muscular atrophy, multiple sclerosis), immunodeficiency (e.g. HIV), diabetes, alopecia and aging, in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said chronic disorder.
In another embodiment, the invention encompasses a method of treating a caspase-3 mediated disease in a mammalian patient in need of such treatment, comprising administering to said patient a compound of formula I in an amount effective to treat said caspase-3 mediated disease.
In particular, these compounds are preferably useful to treat, prevent or ameliorate in mammals and especially in humans, diseases including but not limited to:
cardiac and cerebral ischemia/reperfusion injury (e.g. stroke)
type I diabetes
immune deficiency syndrome (including AIDS)
cerebral and spinal cord trauma injury
organ damage during transplantation
alopecia
aging
sepsis
bacterial meningitis
Parkinson""s disease
Alzheimer""s disease
Down""s syndrome
spinal muscular atrophy
multiple sclerosis
neurodegenerative disorders.
The compound is adminstered to a mammalian patient in need of such treatment or prevention an amount of a compound as described herein that is effective to treat or prevent the disease or condition.
The compounds described typically contain asymmetric centers and may thus give rise to diastereomers and optical isomers. The present invention is meant to comprehend such possible diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
The pharmaceutical compositions of the present invention comprise a compound of formula I as an active ingredient or a pharmaceutically acceptable salt thereof in combination with a pharmaceutically acceptable carrier, and optionally other therapeutic ingredients. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d refers to salts prepared from pharmaceutically acceptable bases including inorganic bases and organic bases. Representative salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, ammonium, potassium, sodium, zinc and the like. Particularly preferred are the calcium, magnesium, potassium, and sodium salts. Representative salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, N,Nxe2x80x2-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine and the like.
When the compound of the present invention is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Examples of such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.
In the discussion of methods of treatment which follows, reference to the compounds of formula I are meant to also include the pharmaceutically acceptable salts.
The ability of the compounds of formula I to inhibit caspase-3 make them useful research tools in the field of apoptosis.
The magnitude of therapeutic dose of a compound of formula I will, of course, vary with the nature of the severity of the condition to be treated and with the particular compound of formula I and its route of administration and vary upon the clinician""s judgement. It will also vary according to the age, weight and response of the individual patient. An effective dosage amount of the active component can thus be determined by the clinician after a consideration of all the criteria and using is best judgement on the patient""s behalf. A representative dose will range from 0.001 mpk/d to about 100 mpk/d.
An ophthalmic preparations for ocular administration comprising 0.001-1% by weight solutions or suspensions of the compounds of formula I in an acceptable ophthalmic formulation may be used.
Any suitable route of administration may be employed for providing an effective dosage of a compound of the present invention. For example, oral, parenteral and topical may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like.
The compositions include compositions suitable for oral, parenteral and ocular (ophthalmic). They may be conveniently presented in unit dosage form and prepared by any of the methods well-known in the art of pharmacy.
In practical use, the compounds of formula I can be combined as the active ingredient in intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of preparation desired for administration. In preparing the compositions for oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, alcohols, oils, flavoring agents, preservatives, coloring agents and the like in the case of oral liquid preparations, such as, for example, suspensions, elixirs and solutions; or carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case or oral solid preparations such as, for example, powders, capsules and tablets, with the solid oral preparations being preferred over the liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit form in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques.
Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion or a water-in-oil emulsion. Such compositions may be prepared by any of the methods of pharmacy but all methods include the step of bringing into active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine, the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. For example, each dosage unit may contain from about 0.01 mg to about 1.0 g of the active ingredient.
Compounds of the present invention are conveniently prepared using the procedures described generally below and more explicitly described in the Example section thereafter. 
Bromomethyl ketone 1 is prepared as illustrated in Scheme 1. Reaction of N-fluorenylmethyloxycarbonyl-L-aspartic acid xcex2-tert-butyl ester (Fmoc-L-Asp (OtBu)xe2x80x94OH) (2) (Novabiochem) with iso-butyl chloroformate (IBCF) followed by treating the reaction mixture with an excess of diazomethane yields the diazomethylketone intermediate 3. This intermediate is subjected in situ to a 1:1 mixture of AcOH and 45% aqueous hydrobromic acid (HBr) to give compound 2 as a white powder.
The semicarbazide resin A is prepared according to Scheme 2. Treatment of compound 4 (Webb et al, J. Am. Chem. Soc. 114, 3156 (1992)) with a commercial amino-Merrifield resin in the presence of EDCI and HOBT in dichloromethane followed by removal of the Boc group with trifluoroacetic acid (TFA) in dichloromethane afforded resin A. 
The general procedure for the solid phase synthesis of compound of general structure Ia incorporating a sulfide P1xe2x80x2 side chain, a P1xe2x80x2 carboxylate side chain and a phenoxide side chain is illustrated in Scheme 3.
Bromomethyl ketone 1 is mixed with resin A in THF in the presence of AcOH overnight to furnish resin B. Nucleophilic displacement with an appropriate nucleophile in the presence of suitable bases followed by deprotection of the Fmoc protecting group using piperidine in DMF to give resin C as shown. Resin C is first reacted with pyrazinone acids of general structure II using O-(7-Azabenzotriazol-1-yl)N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate as the activating agent and DIEA as the base, and the resultant resin is treated with a cocktail of TFA and water (9/1, v/v) to furnish the final Product Ia in which RXCH2 represents R3. 
The general scheme for solution phase synthesis of pyrazinone derivatives Ib containing a P1xe2x80x2 amino, a P1xe2x80x2 carboxylate, a P1xe2x80x2 sulfide or a P1xe2x80x2 phenoxide is illustrated in Scheme 4. 
Appropriate pyrazinone acid II is first reacted with xcex2-tbutyl aspartic acid methyl ester hydrochloride (5) in the presence of HATU/DIEA in DMF to give structure 6. 6 is then carefully hydrolyzed with LiOH in THF/H2O and acidified. The resultant acid is treated with IBCF in the presence of NMM in THF and the mixed anhydride is reacted in situ with diazomethane in ether/THF. The diazo intermediate is directly treated with a mixture of 1:1 (v/v) 45%HBr/AcOH to yield the bromomethyl ketone 7. 7 is processed to the final product Ib, wherein Rxe2x80x2XCH2 represents R3, by first reacting with a suitable nucleophile in the presence of appropriate bases and then with a solution of TFA in dichloromethane.
Alternatively as shown in Scheme 5, 6 is carefully hydrolyzed with LiOH in THF/H2O and acidified. The resultant acid is treated with IBCF in the presence of NMM in THF and the mixed anhydride is reduced with NaBH4 to give the corresponding alcohol which is oxidized under the Dess-Martin oxidation conditions to afford aldehydes of general structure Ic. Reaction of Ic with an appropriate oganometallic reagent Rxe2x80x3M followed by oxidation affords ketones of general structure Id wherein Rxe2x80x3 represents R3. 
A general protocol for making the pyrazinone core structure II is illustrated in Scheme 6. 
An appropriate amino ester P-1 wherein R11 is benzyl, methyl, ethyl, propyl, isopropyl or another suitable protecting group is first reacted with ethyl oxalyl chloride in dichloromethane in the presence of triethylamine to give product P-2. The reaction of P-2 with a suitable amino alcohol P-8 (R2 is hydrogen or alkyl) affords alcohol P-3, which is oxidized to the corresponding ketone P-4. Treatment of P-4 with trifluoroacetic acid (TFA) and trifluoroacetic anhydride (TFAA) in acetic acid at approximately 110xc2x0 C. furnishes the cyclized product P-5, which is reacted with phosphorus oxybromide (POBr3) to yield the corresponding bromide P-6.
Reaction of bromide P-6 with an appropriate amine R1xe2x80x94(CRcRd)nxe2x80x94NH2 in ethanol at reflux temperature gives ester P-7 which is hydrolyzed to afford the desired acid II. When n is 0, the reaction may require the presence of a base, such as a hydride base.

Step 1. A mixture of succinic acid mono-methyl ester (5.28 g), DMAP (4.88 g), methylamidoxime (1.1 eq) and EDCI (1.2 eq) in DME was heated to 95-100xc2x0 C. for three days and cooled to room temperature. The mixture was then partitioned between ethyl acetate and 1N HCl and the organic phase was washed with brine, dried, filtered and concentrated. The residue was purified by chromatography to afford compound 20 (4.2 g) as a colorless oil. 1H NMR (400 MHz, acetone-d6): xcex4 3.62 (s, 3H), 3.13 (t, 2H), 2.86 (t, 2H), 2.27 (s, 3H).
The methyl ester in 20 was hydrolyzed as follow: to a solution of 20 (4.2 g) in ethanol (100 mL) and water (35 mL) was added LiOH monohydrate (2.3 g) and the mixture was stirred for 2 hours and then acidified with 1N HCl. The whole mixture was concentrated in vacuo to approximately 15 mL and then extracted with ethyl acetate (3xc3x97). The extracts were combined, washed with brine, dried, filtered and concentrated. The residue was precipitated from ether/hexanes to yield acid 21 (3.6 g) as a white powder. 1H NMR (400 MHz, acetone-d6): xcex4 3.12 (t, 2H), 2.88 (t, 2H), 2.27 (s, 3H).
To a solution of acid 21 (500 mg) in t-butyl alcohol was added diphenyl phosphorus azide (0.76 mL) and triethylamine (0.94 mL) and the mixture was heated to reflux overnight and concentrated. The residue was purified by flash chromatography. Eluting with 5% (v) methanol in dichloromethane gave the desired product 22. 1H NMR (400 MHz, acetone-d6): xcex4 6.19 (br s, 1H), 3.51 (q, 2H), 3.05 (t, 2H), 2.30 (s, 3H), 1.39 (s, 9H). This compound was then treated with 30% (v) TFA in dichloromethane for I hour and concentrated to give the TFA salt of amine 23 (400 mg). 1H NMR (400 MHz, acetone-d6): xcex4 4.40 (t, 2H), 3.51 (t, 2H), 2.29 (s, 3H). This salt was first treated with Amberlite IRA-96(copyright) to remove the trifluoroacetic acid and then processed to the final compound as described.